A paramagetic derivative (spin label) of cortisol will be synthesized and used as a probe to investigate several aspects of the binding of cortisol (and related corticosteroids) to corticosteroid- binding globulin (CBG). Initial experiments will focus on determining the extent to which the spin label binds to CBG (relative to cortisol). If cortisol and the spin label are found to have essentially equal binding affinities, then use of the spin label should provide a simple and convenient method of assaying for the CBG level in blood serum; if such is the case this assay method will be investigated in detail. This method would involve the addition of a measured amount of spin label to a serum sample and the use of esr spectroscopy to determine the amount of bound (toCBG) and unbound spin label at equilibrium. The above values will then be used to calculate the total amount of CBG present in the serum sample. The above method will be used to assay serum samples containing varying amounts of CBG in order to establish the utility of the method. Further studies using the spin label will be conducted to determine the relative affinities of several additional corticosteroids toward binding to CBG; the results will be compared with the structure of the corticosteroid molecules to determine how specific structural modifications influence the ability of the steroid molecule to undergo binding to CBG. The possibility of synthesizing additional spin-labeled derivatives of cortisol (which differ in the site at which the paramagnetic nitroxide moiety is attached to the cortisol molecule) will be investigated. If several such derivatives can be synthesized easily, studies will be conducted to determine how the positioning of the paramagnetic nitroxide moiety in the derivative influences the ability of the spin-labeled derivative to bind to CBG.